13-Halo and 13-deoxy derivatives of C-076 compounds

ABSTRACT

Derivatives of the C-076 compounds are disclosed wherein the 13-position is unsubstituted. The compounds are prepared by removing the glycosyl groups on the 13-position of the C-076 compounds isolated from the fermentation broth of Streptomyces avermitilis, followed by halogenation and subsequent removal of the halogen. The disclosed compounds are antiparasitic, anthelmintic, insecticidal and acaricidal agents.

BACKGROUND OF THE INVENTION

C-076 is a series of macrolides with potent antiparasitic activity. Thecompounds are isolated from the fermentation broth of Streptomycesavermitilis and the morphological characteristics of the microorganismas well as the methods employed to isolate the C-076 compounds are fulldescribed in U.S. Patent Application Ser. No. 772,061.

SUMMARY OF THE INVENTION

This invention is concerned with derivatives of the C-076 compounds.Specifically, it is concerned with C-076 derivatives which areunsubstituted at the 13-position. In addition various other positions ofthe C-076 compounds may be substituted. Thus, it is an object of thisinvention to describe the 13-deoxy compounds of this invention. It is afurther object of this invention to describe various derivatives of said13-deoxy-C-076 compounds. A still further object is to describeprocesses for the preparation of such compounds. A still further objectis to describe methods and compositions using such compounds as theactive ingredient in the treatment of parasitic infections. Furtherobjects will be apparent from a reading of the following description.

DESCRIPTION OF THE INVENTION

The C-076 compounds which are the starting materials for the preparationof the instant compounds are best described in the following structuralformula: ##STR1## wherein R is the α-L-oleandrosyl-α-L-oleandrose groupof the structure: ##STR2## and wherein the broken line indicates asingle or double bond; R₁ is hydroxy and present only when said brokenline indicates a single bond;

R₂ is .[.n-propyl.]. .Iadd.isopropyl .Iaddend.or sec-butyl; and

R₃ is methoxy or hydroxy.

With reference to the foregoing structural formula, the individual C-076compounds are identified as follows:

    ______________________________________                                        C-076   R.sub.1    R.sub.2           R.sub.3                                  ______________________________________                                        A1a     Double bond                                                                              sec-butyl         --OCH.sub.3                              A1b     Double bond                                                                               .[.n-propyl.].                                                                          .Iadd.isopropyl.Iaddend.                                                             --OCH.sub.3                              A2a     --OH       sec-butyl         --OCH.sub.3                              A2b     --OH       .[.n-propyl.].                                                                           .Iadd.isopropyl.Iaddend.                                                             --OCH.sub.3                              B1a     Double bond                                                                              sec-butyl         --OH                                     B1b     Double bond                                                                               .[.n-propyl.].                                                                          .Iadd.isopropyl.Iaddend.                                                             --OH                                     B2a     --OH       sec-butyl         --OH                                     B2b     --OH       .[.n-propyl.].                                                                           .Iadd.isopropyl.Iaddend.                                                             --OH                                     ______________________________________                                    

Based on taxonomic studies, the microorganisms capable of producingthese C-076 compounds are of a new species of the genus Streptomyces,which has been named Streptomyces avermitilis. One such culture,isolated from soil is designated MA-4680 in the culture collection ofMerck & Co. Inc., Rahway, N.J. A C-076 producing sample of this culturehas been deposited in the permanent culture collection of theFermentation Section of the Northern Utilization Research Branch, U.S.Department of Agriculture at Peoria, Ill., and has been assigned theaccession number NRRL 8165. A sample of NRRL 8165 has also beendeposited, without restriction as to availability, in the permanentculture collection of the American Type Culture Collection at 12301Parklawn Drive, Rockville, Md. 20852, and has been assigned theaccession number ATCC 31,267.

The above microorganism is illustrative of a strain of Streptomycesavermitilis which can be employed in the production of the C-076compounds. However, such description also embraces mutants of the abovedescribed microorganism. For example, those C-076 producing mutantswhich are obtained by natural selection or those producted by mutatingagents including X-ray irradiation, ultraviolet irradiation, nitrogenmustard or like treatments are also included within the ambit of thisinvention.

One example of such an organism is a strain of Streptomyces avermitilisMA 4848 which was isolated after irradiation with ultraviolet light ofStreptomyces avermitilis MA 4680. A lyophilized tube and a frozen vialof this culture has been deposited in the permanent culture collectionof the American Type Culture Collection, and they have been assigned theaccession numbers 31272 and 31271 respectively. Slightly higherfermentation yields of C-076 have been obtained using this frozen stockas inoculum.

The compounds of the instant invention are derived from the above C-076compounds by removing the α-L-oleandrosyl-α-L-oleandrose group and alsothe hydroxy group at the 13-position that remains after the disaccharideis removed. In addition, other derivatization of the 13-deoxy C-076compounds is possible such as acylation of one or more of the availablehydroxy groups, reduction of the 22,23 double bond, alkylation of thehydroxy groups, substitution of an alkylthio group at the 23-position,and oxidized variations thereof, as well as the 13-halogenatedcompounds, which are intermediates in the preparation of the 13-deoxycompounds.

The compounds of the instant invention have the following structuralformula: ##STR3## wherein the broken line indicates a single or doublebond; R₁ is hydrogen or halogen;

R₂ is hydrogen, methyl or loweralkanoyl;

R₃ is .[.n-propyl.]. .Iadd.isopropyl .Iaddend.or sec-butyl; and

R₄ is present only when the broken line indicates a single bond andrepresents hydrogen, hydroxy, loweralkanoyloxy, loweralkylthio,loweralkylsulfinyl, loweralkylsulfonyl or loweralkoxy;

provided that when R₂ is hydrogen or methyl, the broken line canindicate only a single bond and R₄ is other than hydroxy.

In the instant invention the term "loweralkoxy" is intended to includethose alkoxy groups containing from 1 to 6 carbon atoms in either astraight or branched configuration. Examples are methoxy, ethoxy,propoxy, iso-propoxy, butoxy, tert-butoxy, pentoxy, hexoxy and the like.

The terms "loweralkylthio," "loweralkylsulfinyl" and"loweralkylsulfonyl" are intended to include those thio, sulfinyl, andsulfonyl groups which contain a lower alkyl group of from 1 to 6 carbonatoms in either a straight or branched chain. Examples are methyl,ethyl, propyl, iso-propyl, butyl, sec-butyl, pentyl, hexyl and the like.

The terms "loweralkanoyl" or "loweralkanoyloxy" are intended to includethose alkanoyl groups or those alkanoyloxy groups which contain from 2to 6 carbon atoms in either a straight or branched configuration.Examples are acetyl, propionyl, butyryl, pentanoyl, hexanoyl, pivaloyland the like.

The term "halogen" or "halo" is intended to include the halogen atoms offluorine, chlorine, bromine and iodine.

The compounds of the instant invention are prepared by a series ofreactions which converts the C-076 starting materials from a13-disaccharide series of compounds to the aglycone compound(13-position is hydroxy) followed by the conversion of the 13-hydroxygroup to the 13-halogen and 13-deoxy groups. In addition, the R₂ and R₄substituent groups and the 22,23-unsaturation are reacted to form othersubstituents.

As is readily apparent from an analysis of the structure of the C-076starting materials, there are five unsaturations in the 1-series ofcompounds. An object of the instant invention is to reduce the22,23-double bond while not affecting the remaining four unsaturationsor any other functional group present on the molecule. It is necessaryto select a specific catalyst for the hydrogenation, one that willselectively hydrogenate the least hindered from among a series ofunsaturations. The preferred catalyst for such a selective hydrogenationprocedure is one having the formula:

    [(R.sub.5).sub.3 P].sub.3 RhX

wherein R₅ is loweralkyl, phenyl, or loweralkyl substituted phenyl and Xis a halogen.

In the preferred catalyst R₅ is phenyl and X is chlorine, that is thecompound tris(triphenylphosphine)rhodium(I)chloride, which is also knownas Wilkinson's homogeneous catalyst.

The reaction is carried out using a catalytic amount of the catalyst.The amount of catalyst is not critical and from 0.05 to 0.5 moles of thecatalyst for each mole of starting material have been successfullyemployed. Molar ratios in the range of 0.25 to 0.40 are preferred.

The hydrogenation is carried out in a hydrogen atmosphere which may beeither at atmospheric pressure or up to about 4 atmospheres pressure ina standard laboratory hydrogenation apparatus. A solvent is employed todissolve both the starting materials and the catalyst. Preferredsolvents are hydrocarbon solvents such as benzene, toluene, petroleumether and other alkane hydrocarbons. The reaction is complete when thecalculated amount of hydrogen has been taken up by the reaction. Thiswill generally require from about 16 to 48 hours. The reaction may becarried out at from room temperature to about 75° C., however, roomtemperature is preferred. The hydrogenation products are isolated andpurified by techniques known to those skilled in the art.

Other reactions may be carried out on the C-076 starting materials or onthe hydrogenated products to prepare other compounds of this invention.While it is possible to complete other reactions on the C-076 startingmaterial and have the hydrogenation step as the final reaction, it ispreferred to carry out the hydrogenation step before the reactions atthe 5- or 13-position. Because the 22,23-double bond is somewhatsusceptible to electrophilic addition, reaction conditions for removingthe sugar groups or acylating the hydroxy groups must be carefullycontrolled if the 22,23-double bond is present. If the 22,23-double bondis hydrogenated first, the other reactions are rendered more facile.

The acylated compounds at the 5- and 23-positions (R₂ or R₄ asloweralkanoyl) are prepared using acylation techniques in which thereaction conditions will vary, depending upon the reactivity of thehydroxy group being acylated. Where there is more than one hydroxy groupto be acylated, different reaction conditions are employed to minimizethe formation of mixtures.

The preferred acylation reagents employed are generally theloweralkanoyl halide, preferably the chloride, or the loweralkanoylanhydride.

In the case of reactions carried out with the halide reagents, it isoften advantageous to include in the reaction mixture a basic compoundcapable of reacting with and neutralizing the hydrogen halide which isliberated during the course of the reaction. Tertiary amines arepreferred such as triethylamine, pyridine, 4-dimethylamino pyridine,diisopropyl ethylamine and the like. The basic compound is required inequimolar amounts relative to the number of moles of hydrogen halidebeing liberated, however excess amounts, even using the basic compoundas a solvent, are not detrimental.

In the case of the A1 aglycone compounds, there are no hydroxy groupswhich may be acylated.

The A2 compounds have a single available hydroxy group at the23-position capable of being acylated.

The 23-acyl compound may be prepared by heating the reaction mixture atfrom about room temperature to 100° C. for from 1 to 24 hours.

The B1 compounds also have a single available hydroxy group: at the5-position. The reaction with the acylating agent is carried out inpyridine from about 0° C. to room temperature for from 4 to 24 hours. Torecover the acylated compounds, the reaction mixture is eluted through achromatographic column or a preparative layer chromatographic plate ofalumina or silica gel and the purified compounds are readily isolated.In addition, other techniques, such as high pressure liquidchromatography, may be employed.

The B2 compounds have two hydroxy groups available for substitution: and5-and 23-positions. the 5,23-diacyl compound may be prepared by carryingout the reaction at from room temperature to 100° C. for from 1-24hours. The 5-acyl compound may be prepared by carrying out the reactionat from about 0° C. to room temperature for from 4-24 hours. To preparethe 23-acyl compound, the 5,23-diacyl compound is hydrolyzed with anaqueous base such as aqueous sodium hydroxide, at about room temperaturefor from 1 to 24 hours. The 5-acyl group will be hydrolyzed, leaving the23-monoacyl compound.

The above described acyl compounds are isolated from the reactionmixture using techniques known to those skilled in this art.

The compounds where the 23-substituent (R₄) is loweralkoxy orloweralkylthio are prepared from the 1-series of compounds (thecompounds with a 22,23-unsaturation). This unsaturation is more readilysusceptable to electrophilic addition than the other unsaturations inthe molecule, thus by monitoring reaction conditions carefully, thereaction can be made fairly specific.

The reaction is carried out in the presence of an acid and aloweralkanol or a loweralkylthiol. The reaction may be carried out in aninert, aprotic solvent such as dioxane, tetrahydrofuran, ether and thelike or if the alcohol or thiol is available in sufficient quantities,then said alcohol or thiol may be used in large excess and the inertsolvent dispensed with. Suitable acids are sulfuric, hydrohalic,phosphoric, trifluoroacetic, trifluoromethanesulfonic, and the like. Thepreferred hydrohalic acid is hydrochloric or hydrobromic. The mostpreferred acid is sulfuric acid. The acid is present in the reactionmixture at from about 0.1 to 10% by weight. The reaction is completegenerally at from 0°-50° C. for from 2 to 24 hours. It is preferred tostir the reaction mixture overnight at room temperature.

Occasionally, to a small extent, there may be found some 22-additionproducts in the reaction mixture. This will be a minor side product,since the 23-substituent is the thermodynamically preferred compound,and the impurity is readily removed by chromatographic separation.

The 23-loweralkylthio substituent is oxidized to the23-loweralkylsulfinyl and 23-loweralkylsulfonyl group with a mildoxidizing agent. The preferred oxidizing agent is m-chloroperbenzoicacid and the reaction is generally carried out in a solvent inert tooxidation. Halogenated hydrocarbons such as methylene chloride orchloroform are suitable. To prepare the sulfoxide a single molarequivalent of the oxidizing agent is employed and the reaction iscomplete in about 5 minutes to 1 hour at from -20° C. to roomtemperature. To prepare the sulfone two equivalents of the oxidizingagent are used and the reaction is complete in about 1-24 hours at from0° C. to room temperature. The products are isolated using techniquesknown to those skilled in this art.

The 13-position substituents (R₁ =halogen, hydrogen) are prepared fromthe C-076 starting materials as described hereinbelow. The reaction atthe 13-position can generally be carried either before or after theother above described reactions.

The series of reactions at the 13-position commences with the removal ofthe α-L-oleandrosyl-α-L-oleandrose side chain which is found in theC-076 starting materials. This reaction produces what is identified asthe "C-076 aglycone" compounds characterized by having a hydroxy groupat the 13-position. The C-076 aglycone compounds are then halogenatedwith a suitably reactive benzenesulfonyl chloride or bromide in thepresence of a base to produce the "13-deoxy-13-halo-C-076-aglycone"compounds. The halogen is then removed in a reaction with atrialkyltinhydride to produce the "13-deoxy-C-076 aglycone compounds."

The reaction conditions which are generally applicable to thepreparation of C-076 aglycone involve dissolving the C-076 compound inan aqueous non-nucleophilic organic solvent, miscible with water,preferably dioxane, tetrahydrofuran, dimethoxyethane, dimethylformamide, bis-2-methoxyethyl ether and the like, in which the waterconcentration is from 0.1 to 20% by volume. Acid is added to the aqueousorganic solvent to the extent of 1.0 to 10% by volume. The reactionmixture is generally stirred at about 20°-40° C., preferably at roomtemperature, for from 6 to 24 hours. The products are isolated, andmixtures are separated by techniques such as column, thin layer,preparative layer and high pressure liquid chromatography, and otherknown techniques.

The acids which may be employed in the above process include mineralacids and organic acids such as sulfuric, hydrohalic, phosphoric,trifluoroacetic, trifluoromethanesulfonic and the like. The hydrohalicacids are preferably hydrochloric or hydrobromic. The preferred acid inthe above process in sulfuric acid.

A further procedure for the preparation of the aglycone compounds isapplicable to all of the C-076 compounds, however, it is preferred foruse on the compounds wherein the broken line indicates a single bond,since some degree of addition to the 22,23-double bond is noticed inthose compounds with the 22,23-unsaturation. The procedure for thepreparation of the aglycone, 1% sulfuric acid, by volume, in methanol atfrom 20°-40° C., preferably room temperature, for from 6-24 hours hasbeen found to be appropriate.

The other acids listed above may also be employed for this process, atapproximately the concentration employed for sulfuric acid.

The above described compounds are isolated from the reaction mixture andmixtures of compounds are separated using techniques known to thoseskilled in this art, and in particular the chromatographic techniquesdescribed above.

The "C-076 aglycone" thus produced is then halogenated to produce the13-deoxy-13-halo-C-076 aglycone. The halogenation is most readilycarried out in the presence of a sufficiently reactivebenzenesulfonylhalide compound in the presence of a base. The presenceof electron withdrawing substituents on the benzenesulfonylhalide isadvantageous and o-nitro substitution is preferred. The reaction iscarried out in a non-protic inert solvent such as a halogenated alkylcompound, preferably methylene chloride or chloroform. The reactants arecombined slowly at an initial temperature of from -25° to +10° C. tocontrol any initial exothermic reactions and is maintained at thistemperature for up to 2 hours. The reaction temperature is then raisedto from about room temperature to the reflux temperature of the reactionmixture for from 10 minutes to 6 hours. It is necessary to carry out thereaction in the presence of a base, preferably an organic amine. It hasbeen found to be preferable to employ the combination of a4-diloweralkylamino pyridine and trialkylamine. It is most preferred toemploy 4-dimethylamino pyridine and diisopropylethylamine as bases forthe foregoing reactions. The 13-deoxy-13-halo-C-076 aglycone compoundsare isolated by procedures known to those skilled in this art.

In order to avoid unwanted side-reactions, it is important that, inthose C-076 compounds with a hydroxy group at the 5-position (theB-series of compounds), and to a lesser extent, the 23-hydroxy group ofthe 2-series of compounds, said hydroxy groups be protected. Theprotecting group is ideally one which may be readily synthesized, willnot be affected by the reaction to alter the 13-position substituent,and may be readily removed wthout affecting any other function of themolecule. One preferred type of protecting group for the C-076 type ofmolecule is the trisubstituted silyl group, preferably a trialkylsilylgroup. One preferred example is the tert-butyldimethylsilyl group. Thereaction is carried out by reacting the hydroxy compound with theappropriately substituted silyl halide, preferably the silyl chloride,in an aprotic polar solvent such as dimethylformamide. Imidazole isadded as a catalyst. The reaction is complete in from 1/2 to 24 hours atfrom 0°-25° C. For the 5-position hydroxy group of the reaction iscomplete in about 1/2 to 3 hours at from 0° C. to room temperature. Thesilylation reaction is much slower at the 23-position hydroxy group (the2-series of compounds), then at the 5-position hydroxy group, andprotection is generally not necessary. However, if it is desired toprotect the 23-hydroxy group, the reaction will be complete in about 5to 24 hours at from about room temperature to 75° C. This reaction isselective to the 5- and 23-positions under the conditions abovedescribed, and very little, if any, silylation is observed at the13-position.

The silyl group may be removed after the 13-halogenation of the reactionmay be deferred until after the 13-halo group is removed. The silylgroup or groups are removed by stirring the silyl compound is methanolcatalyzed by a catalytic amount of an acid, preferably a sulfonic acidsuch as p-toluenesulfonic acid. The reaction is complete in about 1 to12 hours at from 0° to 50° C.

The 13-deoxy-13-halo-C-076 aglycone which may or may not have the silylgroups protecting the 5- and 23-hydroxy groups is then reduced to formthe 13-deoxy-C-076 aglycone. The preferred reducing agent is one thatwill selectively remove the 13-halo group but will leave the remainderof the molecule untouched. One such reducing agent is atrialkyltinhydride, preferably tributyltinhydride. In addition it ispreferable to include in the reaction mixture a free radical initiatorsince it is believed that the reaction proceeds through a free radicalmechanism (not wishing to be bound by theory, however, other possiblemechanisms are not excluded). Acceptable free radical initiators arevaried and include peroxides, such as dibenzoyl peroxides; thiols in thepresence of air; azodialkylnitriles such as azobisisobutyronitrile;ultraviolet light; heat and the like. The reaction conditions will varydepending upon the type of free radical initiator which is employed. Forchemical initiators the reaction is complete in about 1 to 6 hours atfrom 60°-120° C. The preferred reaction temperature is about 85° C. Ifheat is the initiating agent, higher temperatures are required, about100°-200° C. for from 1-6 hours. If ultraviolet light is employed, lowertemperatures are preferred. Generally the reaction will be complete infrom 1-6 hours at -25° to 50° C. in the presence of ultraviolet light.The trialkyltinhydride reaction is generally carried out with no solventunder a a blanket of nitrogen or other inert gas. The tin hydridecompound is used in excess and becomes the solvent. If desired, however,an inert solvent such as benzene, toluene, xylene and the like could beemployed. For obvious reasons, halogenated solvents cannot be employed.The products are isolated using procedures known to those skilled inthis art.

Except for the csse of the 22,23-hydrogenation reaction and thesilylation reaction above mentioned, there is no requirement that theabove reactions be carried out in any particular order. No conflictingreactions, save for the above exceptions, are found in the foregoingseries of reactions and a reaction at one particular position will notaffect any substituent groups at another reaction.

The novel 13-halo- and 13-deoxy-C-076 compounds of this invention havesignificant parasiticidal activity as anthelmintics, ectoparasiticides,insecticides and acaricides, in human and animal health and inagriculture.

The disease or group of diseases described generally as helminthiasis isdue to infection of an animal host with parasitic worms known ashelminths. Helminthiasis is a prevalent and serious economic problem indomesticated animals such as swine, sheep, horses, cattle, goats, dogs,cats and poultry. Among the helminths, the group of worms described asnematodes causes widespread and often times serious infection in variousspecies of animals. The most common genera of nematodes infecting theanimals referred to above are Haemonchus, Trichostrongylus, Ostertagia,Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis,Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris andParascaris. Certain of these, such as Nematodirus, Cooperia, andOesphagostomum attack primarily the intestinal tract while others, suchas Haemonchus and Ostertagia, are more prevalent in the stomach whilestill others such as Dictyocaulus are found in the lungs. Still otherparasites may be located in other tissues and organs of the body such asthe heart and blood vessels, subcutaneous and lymphatic tissue and thelike. The parasitic infections known as helminthiases lead to anemia,malnutrition, weakness, weight loss, severe damage to the walls of theintestinal tract and other tissues and organs and, if left untreated,may result in death of the infected host. The 13-halo-and 13-deoxy-C-076compounds of this invention have unexpectedly high activity againstthese parasites, and in addition are also active against Dirofilaria indogs, Nematospiroides, Syphacia, Aspiculuris in rodents, arthropodectoparasites of animals and birds such as ticks, mites, lice, fleas,blowfly, in sheep Lucillia sp., biting insects and such migratingdiperous larvae as Hypoderma sp. in cattle, Gastrophilus in horses, andCuterebra sp. in rodents.

The instant compounds are also useful against parasites which infecthumans. The most common genera of parasites of the gastro-intestinaltract of man are Ancylostoma, Necator, Ascaris, Strongyloides,Trichinella, Capillaria, Trichuris, and Enterobius. Other medicallyimportant genera of parasites which are found in the blood or othertissues and organs outside the gastrointestinal tract are the filiarialworms such as Wuchereria, Brugia, Onchocerca and Loa, Dracunculus andextra intestinal stages of the intestinal worms Strogyloides andTrichinella. The compounds are also of value against arthropodsparasitizing man, biting insects and other dipterous pests causingannoyance to man.

The compounds are also active against household pests such as thecockroach, Blatella sp., clothes moth, Tineola sp., carpet beetle,Attagenus sp., and the housefly Musca domestica.

The compounds are also useful against insect pests of stored grains suchas Tribolium sp., Tenebrio sp. and of agricultural plants such as spidermites, (Tetranychus sp.), aphids, (Acyrthiosiphon sp.); againstmigratory orthopterans such as locusts and immature stages of insectsliving on plant tissue. The compounds are useful as a nematocide for thecontrol of soil nematodes and plant parasites such as Meloidogyne spp.which may be of importance in agriculture.

These compounds may be administered orally in a unit dosage form such asa capsule, bolus or tablet, or as a liquid drench where used as ananthelmintic in mammals. The drench is normally a solution, suspensionor dispersion of the active ingredient usually in water together with asuspending agent such as bentonite and a wetting agent or likeexcipient. Generally, the drenches also contain an antifoaming agent.Drench formulations generally contains from about 0.001 to 0.5% byweight of the active compound. Preferred drench formulations may containfrom 0.01 to 0.1% by weight. The capsules and boluses comprise theactive ingredient admixed with a carrier vehicle such as starch, talc,magnesium stearate, or di-calcium phosphate.

Where it is desired to administer the C-076 derivatives in a dry, solidunit dosage form, capsules, boluses or tablets containing the desiredamount of active compound usually sre employed. These dosage forms areprepared by intimately and uniformly mixing the active ingredient withsuitable finely divided diluents, fillers, disintegrating agents and/orbinders such as starch, lactose, talc magnesium stearate, vegetable gumsand the like. Such unit dosage formulations may be varied widely withrespect to their total weight and content of the antiparasitic agentdepending upon factors such as the type of host animal to be treated,the severity and type of infection and the weight of the host.

When the active compound is to be administered via an animal feedstuff,it is intimately dispersed in the feed or used as a top dressing or inthe form of pellets which may then be added to the finished feed oroptionally fed separately. Alternatively, the antiparasitic compounds ofour invention may be administered to animals parenterally, for example,by intramuscular, intratracheal, or subcutaneous injection in whichevent the active ingredient is dissolved or dispersed in a liquidcarrier vehicle. For parenteral administration, the active material issuitably admixed with an acceptable vehicle, preferably of the vegetableoil variety such as peanut oil, cotton seed oil and the like. Otherparenteral vehicled such as organic preparation using solketal,glycerol, formal and aqueous parenteral formulations are also used. Theactive 13-halo- or 13-deoxy-C-076 compound or compounds are dissolved orsuspended in the parenteral formulation for administration; suchformulations generally contain from 0.005 to 5% by weight of the activecompound.

Although the antiparasitic agents of this invention find their primaryuse in the treatment and/or prevention of helminthiasis, they are alsouseful in the prevention and treatment of diseases caused by otherparasites, for example, arthropod parasites such as ticks, lice, fleas,mites and other biting insects in domesticated animals and poultry. Theyare also effective in treatment of parasitic diseases that occur inother animals including humans. The optimum amount to be employed forbest results will, of course, depend upon the particular compoundemployed, the species of animal to be treated and the type and severityof parasitic infection or infestation. Generally good results areobtained with our novel compounds by the oral administration of fromabout 0.001 to 10 mg. per kg. of animal body weight, such total dosebeing given at one time or in divided doses over a relatively shortperiod of time such as 1-5 days. With the preferred compounds of theinvention, excellent control of such parasites is obtained in animals byadministering from about 0.025 to 0.5 mg. per kg. of body weight in asingle dose. Repeat treatments are given as required to combatre-infections and are dependent upon the species of parasite and thehusbandry techniques being employed. The techniques for administeringthese materials to animals are known to those skilled in the veterinaryfield.

When the compounds described herein are administered as a component ofthe feed of the animals, or dissolved or suspended in the drinkingwater, compositions are provided in which the active compound orcompounds are intimately dispersed in an inert carrier or diluent. Byinert carrier is meant one that will not react with the antiparasiticagent and one that may be administered safely to animals. Preferably, acarrier for feed administration is one that is, or may be, an ingredientof the animal ration.

Suitable compositions include feed premixes or supplements in which theactive ingredient is present in relatively large amounts and which aresuitable for direct feeding to the animal or for addition to the feedeither directly or after an intermediate dilution or blending step.Typical carriers or diluents suitable for such compositions include, forexample, distillers' dried grains, corn meal, citrus meal, fermentationresidues, ground oyster shells, wheat shorts, molasses solubles, corncob meal, edible bean mill feed, soya grits, crushed limestone and thelike. The active 13-halo or 13-deoxy-C-076 compounds are intimatelydispersed throughout the carrier by methods such as grinding, stirring,milling or tumbling. Compositions containing from about 0.005 to 2.0% byweight of the active compound are particularly suitable as feedpremixes. Feed supplements, which are fed directely to the animal,contain from about 0.0002 to 0.3% by weight of the active compounds.

Such supplements are added to the animal feed in an amount to give thefinished feed the concentration of active compound desired for thetreatment and control of parasitic diseases. Although the desiredconcentration of active compound will vary depending upon the factorspreviously mentioned as well as upon the particular C-076 derivativeemployed, the compounds of this invention are usually fed atconcentrations of between 0.00001 to 0.002% in the feed in order toachieve the desired antiparasitic result.

In using the compounds of this invention, the individual 13-halo- and13-deoxy-C-076 components may be prepared and used in that form.Alternatively, mixtures of two or more of the individual 13-halo- and13-deoxy-C-076 components may be used, as well as mixtures of the parentC-076 compounds and the compounds of this invention.

In the isolation of the C-076 compounds, which serve as startingmaterials for the instant processes, from the fermentation broth, thevarious C-076 compounds will be found to have been prepared in unequalamounts. In particular an "a" series compound will be prepared in ahigher proportion than the corresponding "b" series comound. The weightratio of "a" series to the corresponding "b" series is about 85:15 to99:1. The differences between the "a" series and "b" series is constantthroughout the C-076 compounds and consists of an .[.n-butyl group and asec-propyl.]. .Iadd.sec-butyl and isopropyl .Iaddend.group respectivelyat the 25-position. This difference, of course, does not interfere withany of the instant reactions. In particular, it may not be necessary toseparate the "b" components from the related "a" component. Separationof these closely related compounds is generally not practiced since the"b" compound is present only in a very small percent by weight, and thestructural difference has negligible effect on the reaction processesand biological activities.

The C-076 compounds of this invention are also useful in combattingagricultural pests that inflict damage upon crops while they are growingor while in storage. The compounds are applied using known techniques assprays, dusts, emulsions and the like, to the growing or stored crops toeffect protection from such agricultural pests.

The following examples are provided in order that this invention mightbe more fully understood; they are not to be construed as limitative ofthe invention.

The 13-halo- and 13-deoxy-C-076 derivatives prepared in the followingexamples are generally isolated as amorphous solids and not ascrystalline solids. They are characterized analytically using techniquessuch as mass spectrometry, nuclear magnetic resonance, and the like.Being amorphous, the compounds are not characterized by sharp meltingpoints, however, the chromatographic and analytical methods employedindicate that the compounds are pure.

EXAMPLE 1 23-O-t-Butyldimethylsilyl-C-076-A2a-Aglycone

200 Mg. of C-076-A2a-aglycone in 2.4 ml. of dry dimethylformamide iscombined with 133 mg. of imidazole and stirred until all the omponentsare dissolved. 146 Mg. of t-butyldimethylsilylchloride is added and thereaction mixture stirred at room temperature for 24 hours. The reactionmixture is diluted with ether and washed five times with water. Thecombined water washes are extracted with ether and the combined organiclayers washed again with water, followed by a single wash with saturatedsodium chloride solution. The ether layer is concentrated to dryness invacuo affording 340 mg. of a gold colored oil. Preparative layerchromatography of the oil on two plates of silica gel eluting with amixture of 5% tetrahydrofuran and 5% ethanol in methylene chlorideaffords 113.2 mg. of 23-O-t-butyldimethylsilyl-C-076-A2a-aglycone, thestructure of which is confirmed by mass spectrometry, and nuclearmagnetic resonance.

EXAMPLE 223-O-t-Butyldimethylsilyl-13-Chloro-13-Deoxy-C-076-A2a-Aglycone

20 Mg. of 23-O-t-butyldimethylsilyl-C-076-A2a-aglycone is combined with0.7 ml. of a methylene chloride solution containing 15 mg. of4-dimethylaminopyridine and 0.021 ml. (15.5 mg.) ofdiisopropylethylamine. The mixture is cooled in an ice bath and asolution of 0.1 ml. of methylene chloride containing 20 mg. ofo-nitrobenzenesulfonylchloride is added dropwise. The reaction mixtureis stirred for 45 minutes in an ice bath, and for 3 hours at roomtemperature. Ice chips are added to the reaction mixture and stirred.When the ice is melted ether is added to the mixture and the layersseparated. The aqueous layer is again extracted with ether and thecombined organic layers washed twice with water, dried over magnesiumsulfate and evaporated to dryness under a stream of nitrogen affording35 mg. of a gold film. Preparative layer chromatography of the materialon a single silica gel plate eluting with 5% tetrahydrofuran and 5%ethanol in methylene chloride affords 10.1 mg. of23-O-t-butyldimethylsilyl-13-chloro-13-deoxy-C- 076-A2a-Aglycone, thestructure of which is confirmed by mass spectrometry and 300 MHz nuclearmagnetic resonance.

EXAMPLE 3 13-Chloro-13-Deoxy-C-076-A2a-Aglycone

A solution of 10mg. of23-O-t-butyldimethylsilyl-13-deoxy-C-076-A2a-aglycone in 1.0 ml. ofmethanol containing 1% p-toluene sulfonic acid dihydrate is stirred atroom temperature for 5 hours. The reaction mixture is diluted with 25ml. of ethyl acetate, and washed with aqueous sodium bicarbonate andwater. The organic layer is dried and evaporated to dryness in vacuoaffording 13-chloro-13-deoxy-C-076A2a-aglycone.

EXAMPLE 4 13-Chloro-13-Deoxy-C-076-A2a-Aglycone

20 Mg. of C-076-A2a-aglycone is dissolved in 0.7 ml. of methylenechloride containing 16 mg. of 4-dimethylaminopyridine and 16.8 mg.(0.023 ml.) of diisopropylethylamine. The reaction mixture is cooled inan ice bath and 0.1 ml. of methylene chloride containing 21.5 mg. ofo-nitrobenzenesulfonylchloride is added dropwise. The reaction mixtureis stirred in an ice bath for 1 hour, allowed to warm to roomtemperature and stirred for 4 hours. Ice is added and stirred untilmelted. Ether is added and the layers shaken and separated. The aqueouslayer is extracted with ether and the organic layers combined, washedthree times with water, dried over magnesium sulfate and evaporated todryness under a stream of nitrogen affording 40 mg. of a brown film.Preparative layer chromatography on silica gel eluting with 3%tetrahydrofuran and 1% ethanol in methylene chloride affords 4.7 mg. of13-chloro-13-deoxy-C-076-A2a-aglycone, which is identified by a nuclearmagnetic resonance and mass spectrometry.

EXAMPLE 5 13-Deoxy-C-076-A2-a-Aglycone

80 Mg. 13-chloro-13-deoxy-C-076-A2a-aglycone is dissolved in 1.5 ml. oftributyltinhydride and 20 mg. of azobisisobutylronitrile is added. Thereaction is heated under a blanket of nitrogen at 85° C. for 31/2 hours,cooled and placed on a silica gel preparative layer chromatography plateand eluted with chloroform affording 110 mg. of a glass. Repeatedpreparative layer chromatography on silica gel using methylene chloridewith 2% tetrahydrofuran and 0.07% ethanol as eluent affords 70 mg. of awhite glass which is identified by mass spectrometry and mHz nuclearmagnetic resonance as 13-deoxy-C-076-A2a-aglycone.

EXAMPLE 6 5-O-t-Butyldimethylsilyl-C-076-B1a-Aglycone

100 Mg. of C-076-B1a-aglycone is dissolved in 1.2 ml. of anhydrousdimethylformamide and 46 mg. of imidazole is added followed by 50 mg. oft-butyldimethylsilylchloride. The reaction is maintained at 20° C. for30 minutes and diluted with ether. The mixture is washed with water,dried and concentrated in vacuo to a colorless glass. Furtherpurification on a preparative layer chromatography plate eluting with amethylene chloride, tetrahydrofuran mixture affords purified5-O-t-butyldimethylsilyl-C-076-B1a-aglycone.

Following the above procedure, utilizing C-076-B2a-aglycone in place ofC-076-B1a-aglycone, affords 5-O-t-butyldimethylsilyl-C-076-B2a-aglycone.

EXAMPLE 7 5-O-t-Butyldimethylsilyl-13-Deoxy-13-chloro-C-076-B1a-Aglycone

Following the procedure of Example 4 utilizing5-O-t-butyldimethylsilyl-C-076-B1a-aglycone in place ofC-076-A2a-aglycone, there is produced5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B1a-aglycone.

Following the above referenced procedure using5-O-t-butyldimethylsilyl-C-076-B2a-aglycone in place of5-O-t-butyldimethylsilyl-C-076-B1a-aglycone, there is obtained5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B2a-aglycone.

EXAMPLE 8 5-O-t-Butyldimethylsilyl-13-Deoxy-C-076-B1a-Aglycone

Following the procedure of Example 5 utilizing5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B1a-aglycone in placeof 13-chloro-13-deoxy-C-076-A2a-aglycone, there is produced5-O-t-butylmethylsilyl-13-deoxy-C-076-B1a-aglycone.

Following the above referenced procedure using5-O-t-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B2a in place of5-O-butyldimethylsilyl-13-deoxy-13-chloro-C-076-B1a-aglycone, there isproduced 5-O-t-butyldimethylsilyl-13-deoxy-C-076-B2a-aglycone.

EXAMPLE 9 13-Deoxy-C-076-B1a-Aglycone

A solution of 13 mg. of5-O-t-butyldimethylsilyl-13-deoxy-C-076-B1a-aglycone in 1.0 ml. ofmethanol containing 1% p-toluenesulfonic acid dihydrate is stirred at20° C. for 3 hours. The reaction is diluted with 30 ml. of ethylacetate, washed with aqueous sodium bicarbonate solution, and then withwater. The organic layer is dried and evaporated to dryness in vacuo toafford 13-deoxy-C-076-B1a-aglycone as a clear glass.

Following the above procedure, utilizing5-O-t-butyldimethylsilyl-13-deoxy-C-076-B2a-aglycone in place of5-O-t-butyldimethylsilyl-13-deoxy-C-076-B1a-aglycone, there is obtained13-deoxy-C-076-B2a-aglycone.

If the products of Example 7 are hydrolized according to the foregoingprocedure, there will be obtained 13-chloro-13-deoxy-C-076-B1a-aglyconeand 13-chloro-13-deoxy-C-076-B2a-aglycone.

EXAMPLE 10 13-Chloro-13-Deoxy-22,23-Dihydro-C-076-A1a-Aglycone

A solution of 8.2 mg. of 22,23-dihydro-C-076-A1a-aglycone and 0.35 ml.of methylene chloride containing 7.5 mg. of 4-dimethylaminopyridine and10.5 microliters of diisopropylethylamine is cooled to 0° C. and treatedwith 10 mg. of o-nitrobenzenesulfonylchloride. After stirring for 1 hourat 0° C. the reaction is warmed to room temperature for 2 hours. Thereaction mixture is quenched with ice and treated with 2 ml. of ether.The layers are separated and aqueous phase washed twice with 1 ml. ofether. The combined organic layers are washed twice with water, driedover sodium sulfate and evaporated to dryness in vacuo. The product isisolated by preparative layer chromatography on a single silica gelplate eluting with chloroform. Lyophilization of the residue affords 1.3mg. of a white powder identified by mass spectrometry and nuclearmagnetic resonance as13-chloro-13-deoxy-22,23-dihydro-C-076-A1a-aglycone.

EXAMPLE 11 13-Deoxy-22,23-Dihydro-C-076-A1a-Alglycone

A solution of 1.0 mg. of13-chloro-13-deoxy-22,23-dihydro-C-076-A1a-aglycone is dissolved in 0.2ml. of tributyltinhydride containing 0.2 mg. of azobisisobutyronitrileand heated under nitrogen at 85° C. for 31/2 hours. The mixture iscooled and chromatographed on a single silica gel preparative layerchromatography plate eluting with chloroform. The remainingtributyltinhydride and tributyltinchloride move with the solvent frontand the product is found at Rf of about 0.15 to 0.4. This band iscollected and eluted from the silica gel with ethyl acetate. The mixtureis chromatographed on a preparative layer silica gel chromatographyplate eluting with chloroform affording 0.5 mg. of13-deoxy-22,23-dihydro-C-076-A1a-aglycone identified by massspectrometry and nuclear magnetic resonance.

EXAMPLE 12 5-O-t-Butyldimethylsilyl-22,23-Dihydro-C-076-B1a-Aglycone

50 mg. of 22,23-dihydro-C-076-B1a-aglycone is dissolved in 1.1 ml. ofdimethylformamide containing 60 mg. imidazole. While under nitrogen 75mg. of t-butyl-dimethylsilychloride is added and the stoppered mixtureis stirred overnight at room temperature. The reaction is quenched with2 ml. of water after dilution of the reaction mixture 15 ml. of ether.The aqueous phase is separated and extracted with 5 ml. of ether. Thecombined organic phases are washed 5 times with 10 ml. of water, thecombined aqueous washes are extracted with 5 ml. of ether, and thecombined organic phases washed once again with 5 ml. of water. Theorganic layer is dried over magnesium sulfate and evaporated to drynessin vacuo to an oil. The oil is chromatographed on 2 silica gelpreparative layer chromatography plates eluting twice with methylenechloride. The slowest moving and most intense band is collected andwashed from the silica gel with ethyl acetate. Lyophilization frombenzene affords 36.3 mg. of a white powder identified by nuclearmagnetic resonance and mass spectrometry as5-O-t-butyldimethylsilyl-22,23-dihydro-C-76-B1a-aglycone.

EXAMPLE 1313-Chloro-13-Deoxy-5-O-t-Butyldimethylsilyl-22,23-Dihydro-C-076-B1a-Aglycon

A solution of 35.5 mg. of5-O-t-butyldimethylsilyl-22,23-dihydro-C-076-B1a-aglycone in 2.6 ml. ofmethylene chloride containing 56 mg. of 4-dimethylaminopyridine pyridineand 78 microliters (59 mg.) of diisopropylethylamine is cooled to 0° C.and treated with 75 mg. of o-nitrobenzenesulfonyl chloride. The reactionmixture is stirred for 1 hour at 0° C., allowed to warm to roomtemperature and stirred for 3 hours. 3 Ml. of crushed ice is added tothe reaction mixture followed by 4 ml. of ether. The layers areseparated and the aqueous phase washed with 4 ml. of ether and thecombined organic phases washed twice with 5 ml. of water. The organiclayer is dried over sodium sulfate and evaporated to dryness in vacuo.Benzene is added to the residue and azeotroped away. The product isisolated by preparative layer chromatography eluting with a 1:2 mixtureof petroleum ether (b.p. 30° to 60° C.) and chloroform to afford 5.4 mg.of13-chloro-13-deoxy-5-O-t-butyldimethylsilyl-22,23-dihydro-C-076-B1a-Aglyconeidentified by mass spectrometry and nuclear magnetic resonance.

EXAMPLE 1413-Deoxy-5-O-t-Butyldimethylsilyl-22,23-Dihydro-C-076-B1a-Aglycone

A solution of 13.2 mg. of13-chloro-13-deoxy-5-O-butyldimethylsilyl-22,23-dihydro-C-076-B1a-aglyconeis combined with 0.7 ml. of tributyltinhydride and 2.0 mg. ofazobisisobutyronitrile and heated to 85° C. for 31/2 hours under ablanket of nitrogen. The reaction mixture is cooled and dissolved in 1.5ml. of methylene chloride and filtered through a column of silica geleluting with methylene chloride. The tributyltinhydride andtributyltinchloride pass through the column upon washing with 250 ml. ofmethylene chloride and the product remains on the column. The solvent ischanged to ethyl acetate and the product eluted at the solvent front.The ethyl acetate solution is concentrated to an oil and the productpurified by preparative layer chromatography on silica gel plateseluting with a 1:1 mixture of petroleum ether (b.p. 30° to 60° C.) andmethylene chloride to afford, after lyophilization from benzene, 8.2 mg.of 13-deoxy-5-O-t-butyldimethylsilyl-22,23-dihydro-C-076-B1a-aglyconewhich is identified by mass spectrometry and nuclear magnetic resonance.

EXAMPLE 15 13-Deoxy-22,23-Dihydro-C-076-B1a-Aglycone

A solution of 6.9 mg. of13-deoxy-5-O-t-butyldimethylsilyl-22,23-dihydro-C-076-B1a-aglycone in0.6 ml. of 1% p-toluenesulfonic acid in methanol is stirred for 3 hoursat room temperature. The reaction is quenched with 5 ml. of ether and 1ml. of saturated aqueous potassium bicarbonate. The layers are separatedand the aqueous phase washed with 2 ml. of ether and the combinedorganic phases washed with water, dried over sodium sulfate andevaporated to dryness in vacuo. The oil is chromatographed on a singlesilica gel plate eluting with a 2:1 mixture of methylene chloride andpetroleum ether (b.p. 30° to 60° C.). After lyophilization there remains4.5 mg. of 13-deoxy-22,23-dihydro-C-076-B1-a-aglycone identified by massspectrometry and nuclear magnetic resonance.

EXAMPLE 16 13-Deoxy-23-O-t-butyldimethylsilyl-C-076-A2a-aglycone

1 Mg. of 13-chloro-13-deoxy-23-O-t-butyldimethylsilyl-C-076-A2a-aglyconeis dissolved in 50 microliters of toluene and 100 microliters oftributyltinhydride and 200 micrograms of azobisisobutyronitrile andheated at 60° C. for 4 hours. The product is isolated by directchromatography on a preparative layer silica gel chromatography plateeluting with 1.5% tetrahydrofuran in chloroform affording13-deoxy-23-O-t-butyldimethylsilyl-C-076-A2a-aglycone which isidentified by mass spectrometry.

EXAMPLE 17 5-O-Acetyl-13-Chloro-13-Deoxy-C-076-B1a-Aglycone

25 Mg. of 13-chloro-13-deoxy-C-076-B1a-aglycone is dissolved in 0.6 ml.of pyridine and 0.3 ml. of acetic anhydride is added. The reaction isstirred at 20° C. overnight. Ice is added to the reaction mixture,allowed to melt, and extracted with ether. The ether layer is washedwith water, dried and concentrated in vacuo. The residue is purified bypreparative layer chromatography on silica gel, eluting with chloroform,and the structure of 5-O-acetyl-13-chloro-13-deoxy-C-076-B1a-aglycone isconfirmed by mass spectrometry and nuclear magnetic resonance.

EXAMPLE 18 5-O-Acetyl-13-Deoxy-C-076-B1a-Aglycone

Following the procedure of Example 17 using 13-deoxy-C-076-B1a-aglyconein place of 13-chloro-13-deoxy-C-076-B1a-aglycone, there is obtained5-O-acetyl-13-deoxy-C-076-B1a aglycone.

If propionic anhydride is employed in place of acetic anhydride ineither of Examples 17 or 18, the analogous 5-O-propionyl compound isobtained.

EXAMPLE 195-O-t-Butyldimethylsilyl-13-Chloro-13-Deoxy-23-O-Acetyl-C-076-B2a-Aglycone

A mixture of 20 mg. of5-O-t-butyldimethylsilyl-13-chloro-13-deoxy-C-076-B2a-aglycone, 0.8 ml.of pyridine and 0.4 ml. of acetic anhydride is heated in an oil bath for2 hours at 100° C. The reaction mixture is cooled, ice is added, allowedto melt, and the precipitate collected by centrifugation. The solidmaterial is dried, dissolved in methylene chloride and chromatographedon a preparative layer silica gel plate. The product is collected,dissolved in benzene and lyophilized affording5-O-t-butyldimethylsilyl-13-chloro-13-deoxy-23-O-acetyl-C-076-B2a-aglyconeas a white fluffy solid.

EXAMPLE 205-O-t-Butyldimethylsilyl-13-Deoxy-23-O-Acetyl-C-076-B2a-Aglycone

Following the procedure of Example 19 using5-O-t-butyldimethylsilyl-13-deoxy-C-076-B2a-aglycone in place of5-O-t-butyldimethylsilyl-13-chloro-13-deoxy-C-076-B2a-aglycone, there isobtained5-O-t-butyldimethylsilyl-13-deoxy-23-O-acetyl-C-076-B2a-aglycone.

EXAMPLE 21 13-Chloro-13-Deoxy-23-O-Acetyl-C-076-B2a-Aglycone

10 Mg. of5-O-t-butyldimethylsilyl-13-chloro-13-deoxy-23-O-acetyl-C-076-B2a-aglyconeis dissolved in 0.5 ml. of methanol containing 1% by weight ofp-toluenesulfonic acid dihydrate, and stirred at room temperature for 3hours. To the reaction mixture is added 5 ml. of ether and the solutionwashed with aqueous sodium bicarbonate solution, dried and concentratedunder a stream of nitrogen to a colorless glass. The glass is furtherpurified by preparative layer chromatography on silica gel eluting withchloroform, and affording pure13-Chloro-13-deoxy-23-O-acetyl-C-076-B2a-aglycone.

EXAMPLE 22 13-Deoxy-23-O-Acetyl-C-076-B2a-Aglycone

Following the procedure of Example 21 employing5-O-t-butyldimethylsilyl-13-deoxy-23-O-acetyl-C-076-B2a-aglycone inplace of5-O-t-butyldimethylsilyl-13-chloro-13-deoxy-23-O-acetyl-C-076-B2a-aglycone,there is obtained 13-deoxy-23-O-acetyl-C-076-B2a-aglycone.

EXAMPLE 23 13-Chloro-13-Deoxy-5,23-Di-O-Acetyl-C-076-B2a-Aglycone

50 Mg. of 13-chloro-13-deoxy-C-076-B2a-aglycone is dissolved in 1 ml. ofpyridine and 0.5 ml. of acetic anhydride is added. The reaction mixtureis heated for 2 hours at 100° C. Upon cooling to room temperature, icewater is added, producing a precipitate which is collected byfiltration. The solid material is further purified by preparative layerchromatography eluting with 2.1 tetrahydrofuran in chloroform affordingpure 13-chloro-13-deoxy-5,23-di-O-acetyl-C-076-B2a-aglycone.

EXAMPLE 24 13-Deoxy-5,23-Di-O-Acetyl-C-076-B2a-Aglycone

Following the procedure of Example 23 using 13-deoxy-C-076-B2a-aglyconein place of 13-chloro-13-deoxy-C-076-B2a-aglycone, there is obtained13-deoxy-5,23-di-O-acetyl-C-076-B2a-aglycone.

EXAMPLE 25 13-Deoxy-22,23-Dihydro-23-n-Butylthio-C-076-A1a-Aglycone

A solution of 100 mg. of 13-deoxy-C-076-A1a-aglycone in a mixture of 9.4ml. of dioxane, 0.5 ml. of n-butanethiol and 0.1 ml. of concentratedsulfuric acid is stirred at 18° C. for 18 hours. The reaction mixture isdiluted with 80 ml. of ether washed with aqueous sodium bicarbonatesolution, dried and concentrated in vacuo to a light glass. The glass isfurther purified on a preparative layer chromatography silica gel plate.The product is identified by mass spectrometry and nuclear magneticresonance as 13-deoxy-22,23-dihydro-23-n-butylthio-C-076-A1a-aglycone.

EXAMPLE 26

13-Chloro-13-Deoxy-22,23-Dihydro-23-n-Butylthio-C-076-A1a Aglycone

Following the procedure of Example 25 employing13-chloro-13-deoxy-C-076-A1a-aglycone in place of13-deoxy-C-076-A1a-aglycone, one obtains13-chloro-13-deoxy-22,23-n-butylthio-C-076-A1a-aglycone.

EXAMPLE 27

Following Example 25 employing equivalent amounts of methanethiol,isopropylthiol and tert-butylthiol, there is obtained13-deoxy-22,23-dihydro-23-methylthio-C-076-A1a-aglycone,13-deoxy-22,23-dihydro-23-isopropylthio-C-076-A1a-aglycone and13-deoxy-22,23-dihydro-23-tertbutylthio-C-076-A1a-aglycone.

EXAMPLE 28 13-Deoxy-22,23-Dihydro-23-n-Butylsulfinyl-C-076-A1a-Aglycone

A solution of 67 mg. (0.1 mmoles) of13-deoxy-22,23-dihydro-23-n-butylthio-C-076-A1a-aglycone in 1.0 ml. ofchloroform is stirred rapidly at 0° C. A second solution containing 19mg. (0.11 mmoles) of m-chloro perbenzoic acid in 0.5 ml. of chloroformis added dropwise. The reaction mixture is allowed to reach 18° C.,allowed to stand for 2 hours, then diluted with ether, washed withaqueous sodium bicarbonate solution, dried and concentrated under astream of nitrogen to a colorless glass, which is identified as13-deoxy-22,23-dihydro-23-n-butylsulfinyl-C-076-A1a-aglycone.

EXAMPLE 29 13-Deoxy-22,23-Dihydro-23-n-Butylsulfonyl-C-076-A1-Aglycone

Following the procedure of Example 28 employing twice the amount ofm-chloro perbenzoic acid that is 38 mg. (0.22 mmoles) in 1.0 ml. ofHCCl₃, affords13-deoxy-22,23-dihydro-23-n-butylsulfonyl-C-076-A1-aglycone.

EXAMPLE 30 13-Deoxy-22,23-Dihydro-23-Methoxy-C-076-B1a-Aglycone

A solution of 100 mg. of 13-deoxy-C-076-B1a-aglycone cone in a mixtureof 9.9 ml. of methanol and 0.1 ml. of concentrated sulfuric acid ismaintained at 18° C. for 20 hours. The reaction mixture is diluted with100 ml. of ether and washed with aqueous sodium bicarbonate solution.The solution is dried and concentrated in vacuo to a glass. The reactionproduct is further purified on a preparative layer silica gelchromatography plate and identified by nuclear magnetic resonance andmass spectrometry as13-deoxy-22,23-dihydro-23-methoxy-C-076-B1a-aglycone.

EXAMPLE 31

Following the procedure of Example 30 but using ethanol, isopropanol orn-hexanol in place of methanol,13-deoxy-22,23-dihydro-23-ethoxy-C-076-B1a-aglycone;13-deoxy-22,23-dihydro-23-isopropoxy-C-076-B1a-aglycone, and13-deoxy-22,23-dihydro-23-n-hexyloxy-C-076-B1a-aglycone are obtained.

EXAMPLE 3213-Chloro-13-Deoxy-22,23-Dihydro-23-Methoxy-C-076-B1a-Aglycone

Folowing the procedure of Example 30 employing13-chloro-13-deoxy-C-076-B1a-aglycone in place of13-deoxy-C-076-B1a-aglycone there is obtained13-chloro-deoxy-22,23-dihydro-23-methoxy-C-076-B1a-aglycone.

PREPARATION 1 C-076 A1a-Aglycone

100 Mg. of C-076 A1 a is dissolved in 5 ml. of dioxane, stirred andadded at room temperature to a mixture of 0.1 ml. of concentratedsulfuric acid, 1.9 ml. of methanol and 3.0 ml. of dioxane. The reactionmixture is stirred overnight at room temperature. 473 Mg. of solidsodium bicarbonate is added and the mixture stirred for 20 minutes. 3Ml. of water is added and stirred for an additional 10 minutes. Thereaction mixture is concentrated and 40 ml. of chloroform is added andshaken. The aqueous layer is separated and extracted with 5 ml. ofchloroform. The organic layers are combined and washed once with dilutesodium chloride solution, dried over magnesium sulfate and evaporated todryness in vacuo. 1/2 of the residue is placed on 5 perparative layerchromatography silica gel plates and eluted with 2% methanol inchloroform affording 4 bands of material. The remainder of the materialwas run on 2 preparative layer chromatography plates eluting with 2%methanol in chloroform affording 4 band similar to the first series. Thesecond fastest bands are removed from each of the plates, combined,extracted and evaporated to dryness in vacuo, and rechromatographed on apreparative layer chromatography silica gel plate eluting with 3%tetrahydrofuran and chloroform affording 9.4 mg. of a fluffy white solidwhich is identified by mass spectrometry as C-076 -A1a-aglycone.

PREPARATION 2 C-076-A2a-Aglycone

2 G. of C-076 A2a is combined with 40 ml. of a 1% (volume/volume) ofsolution of concentrated sulfuric acid in methanol. The reaction mixtureis stirred at room temperature for 17 hours and diluted with 300 ml. ofchloroform. The mixture is washed once with 30 ml. of saturated sodiumbicarbonate solution, once with 30 ml. saturated sodium chloridesolution, dried over magnesium sulfate and evaporated to dryness invacuo. 5 Ml. of methanol is added to the residue and allowed to stand atroom temperature overnight. Cooling of the mixture in ice causes theslow precipitation of crystals. A supernatant is removed and the solidcrystals washed twice with 1 ml. of cold methanol affording 340 mg. of awhite solid. The mother liquor and washings are evaporated down to avolume of about 2 ml. and allowed to stand affording an additional cropto crystals. 630 Mg. of a white solid is obtained which is combined withthe first batch of crystals and 8 ml. of methanol and evaporated to avolume of 2.5 ml. and allowed to stand for several hours. 910 Mg. of anoff white solid is obtained which mass spectrometry identifies as C-076A2a-aglycone.

PREPARATION 3 C-076-B2a aglycone

2 G. of C-076-B2a is combined with 40 ml. of a 1% solution ofconcentrated sulfuric acid in methanol (volume/volume). The reactionmixture is stirred at room temperature for 17 hours. 300 Ml. ofchloroform is added followed by 30 ml. of an aqueous saturated sodiumbicarbonate solution. The layers are separated and the organic layerwashed with 30 ml. of saturated sodium chloride solution, dried overmagnesium sulfate and evaporated to dryness in vacuo. 5 Ml. of methanolis added to dissolve the residue and the mixture allowed to stand atroom temperature and then cooled in an ice bath, whereuponcrystallization occurred. The supernatant is removed and the residuewashed twice with 1 ml. portions of cold methanol and the solid crystalsdried overnight and then in vacuo at 35° C. affording 1.0 g. of whitecrystals. A second crop is obtained by evaporating the mother liquors toa volume of 2 ml. and allowing to stand overnight at room temperature. 2Ml. of methanol is added and the mixture aged in an ice bath affording140 mg. of a yellow solid. The two solid fractions are combined anddissolved in boiling methanol, about 30 ml. of methanol is required. Thesolution is filtered hot and concentrated to a volume of about 20 ml. invacuo whereupon solids begin to precipitate. The solution is filteredhot and the solid materials washed with methanol affording 340 mg. of awhite solid. The filtrates are boiled down to a volume of about 8 ml.and set aside to crystallize at room temperature affording 433 mg. of awhite solid. Mass spectrometry shows the two fractions to be identicaland to be identified as C-076-B2a-aglycone.

PREPARATION 4 C-076-B1a-Aglycone

100 Mg. of C-076 B1a is dissolved in 2.5 ml. of dioxane and combinedwith 2.5 ml. of a mixture prepared from 0.5 ml. of water, 0.5 ml. ofconcentrated sulfuric acid and 9.0 ml. of dioxane. The reaction mixtureis stirred at room temperature for 17 hours. 50 Ml. of ether and 25 ml.of saturated aqueous sodium bicarbonate is added, the layers separated,and the organic layer washed with water and the water layer extractedwith ether. The organic layers are combined, dried over sodium sulfate,and evaporated to dryness. Benzene is added and the solution againevaporated affording 60 mg. of a yellow oil. The oil is chromatographedon a preparative layer chromatography silica gel plate, eluting with a9:1 mixture of chloroform and tetrahydrofuran affording at an Rf ofabout 0.35, 16 mg. of C-076 B1a-aglycone, which is identified by 300 MNznuclear magnetic resonance.

PREPARATION 5 22,23-Dihydro-C-076-A1a

51.0 Mg. of C-076-A1a and 14.4 mg. oftris(triphenylphosphine)rhodium(I)chloride are combined in 3.5 ml. ofbenzene and hydrogenated for 20 hours at room temperature underatmospheric pressure. The crude reaction mixture is chromatographed on apreparative layer chromatography plate eluting twice with 10%tetrahydrofuran in chloroform. The product is removed from the supportusing ethyl acetate which is evaporated to dryness and the residueanalyzed with 300 MHz nuclear magnetic resonance and mass spectroscopyindicating the preparation of 22,23-dihydro-C-076 A1a.

PREPARATION 6 22,23-Dihydro-C-076 B1a

A solution of 1.007 g. of C-076-B1a, 314 mg. oftris(triphenylphosphine)rhodium(I)chloride and 33 ml. of benzene ishydrogenated for 21 hours at room temperature under 1 atmosphere ofhydrogen pressure. The solvent is removed in vacuo and the residuedissolved in a 1:1 mixture of methylene chloride and ethyl acetate andfiltered. The filtrate is placed on a column of 60 g. of silica geleluting with a 1:1 mixture of methylene chloride and ethyl acetatetaking 10 ml. fractions. Fractions 14-65 are combined and evaporated todryness affording 1.118 g. of a solid material which is indicated byhigh pressure liquid chromatography to be a 60/40 mixture of thehydrogenated product and starting material. The mixture isrehydrogenated in 55 ml. of benzene adding 310 mg. oftris(triphenylphosphine)rhodium(I)chloride and stirring for 21 hours atroom temperature under 1 atmosphere of hydrogen pressure. The solvent isremoved in vacuo and the residue chromatographed on 80 g. of silica gelusing 40:60 mixture of ethyl acetate and methylene chloride as eluant.10 Ml. fractions are taken and the product appears in fractions 26-80.These fractions are combined and evaporated to dryness in vacuoaffording a yellow oil. The oil is dissolved in benzene and lyophilizedaffording a pale yellow powder which is identified as22,23-dihydro-C-076-B1a by mass spectrometry and 300 MHz nuclearmagnetic resonance 0.976 G. of product is obtained.

PREPARATION 7 22,23-Dihydro-C-076-A1a Aglycone

10.1 Mg. of 22,23-dihydro-C-076 A1a is stirred for 20 hours in 1.1 ml.of 1% sulfuric acid in methanol at room temperature. The reactionmixture is treated as in Preparation 6 affording an oil which ispurified by preparative layer chromatography on silica gel eluting with5% tetrahydrofuran in chloroform. The product is removed from thechromatography plate and lyophilized fom benzene affording 4.2 mg. of awhite powder which 300 MHz nuclear magnetic resonance and massspectrometry indicate to be 22,23-dihydro-C-076-A1a aglycone.

PREPARATION 8 22,23-Dihydro-C-076-B1a-Aglycone

0.486 G. of 22,23-dihydro-C-076-B1a is added to a stirred solution 50ml. of 1% sulfuric acid in methanol and the reaction mixture stirred for13 hours at room temperature. The reaction mixture is diluted with 250ml. of methylene chloride and washed with 50 ml. of saturated aqueouspotassium bicarbonate and 50 ml. of water. The aqueous layer is washedtwice with 20 ml. portions of methylene chloride and the combinedorganic phases are dried with saturated brine and sodium sulfate andevaporated to dryness in vacuo affording 0.480 g. of a pale yellow foam.The foam is dissolved in 4 ml. of methylene chloride and placed on 4preparative layer chromatography silica gel plates and eluted 4 timeswith 4% tetrahydrofuran and chloroform. The product is recovered fromthe silica gel plates affording an oily residue which is lyophilizedfrom benzene affording 255.8 mg. of a white solid. Traces of methyloleandroside are indicated to be present in the solid material. Thewhite solid is then lyophilized again from benzene and placed under highvacuum for 20 hours to remove the impurity affording22,23-dihydro-C-076-B1a-aglycone.

PREPARATION 9

A 250 ml. baffled Erlenmeyer flask containing 50 ml. of the followingmedium:

    ______________________________________                                        Lactose              2.0%                                                     Distiller's solubles 1.5%                                                     Autolyzed yeast, Ardamine pH                                                                       0.5%                                                     pH-before sterilization                                                                            7.0                                                      ______________________________________                                    

is inoculated with the contents of one frozen vial of Streptomycesavermitilis MA 4848 and incubated on a rotary shaker at 28° C. for 24hours at 150 RPM.

10 Ml. of the above fermentation media is employed to inoculate 500 ml.of the same medium as above in a 2 liter baffled Erlenmeyer flask. Thefermentation media is incubated at 150 RPM on a rotary shaker at 28° C.for 24 hours.

All of the foregoing media is employed to inoculate 467 liters of thefollowing media in a 756 liter stainless steel fermentor:

    ______________________________________                                        Lactose                2.0%                                                   Distiller's solubles   1.5%                                                   Autolyzed yeast, Ardamine pH                                                                         0.5%                                                   Polyglycol 2000        0.32 ml/liter                                          pH-before sterilization                                                                              7.0                                                    ______________________________________                                    

The fermentation media is incubated at 28° C. for 40 hours with an airflow 10 cubic feet per minute and an agitation rate 130 RPM.

230 Liters of the above media is employed to inoculate 4,310 liters ofthe following medium in a 5,670 liter stainless steel fermentor:

    ______________________________________                                        Dextrose               4.5%                                                   Peptonized milk        2.4%                                                   Autolyzed yeast, Ardamine pH                                                                         0.25%                                                  Polyglycol 2000        2.5 ml/liter                                           pH-before sterilization                                                                              7.0                                                    ______________________________________                                    

The fermentation continues for 144 hours at 26° C. with an air flow rateof 54.3 cubic feet per minute and agitation of 120 RPM.

The fermentation media are filtered and teh mycelial filter cake washedwith about 550 liters of water, the filtrate and washings are discarded.The filter cake is agitated with about 1500 liters of acetone for aboutone hour and filtered. The filter cake is washed with a mixture of about150 liters of acetone and 40 liters of deionized water affording about2000 liters of extract.

The foregoing fermentation and extraction is repeated on the same scaleaffording s further 2000 liters of acetone extract which is combinedwith the first extract and evaporated to a volume of about 800 liters.The pH of the concentrate is adjusted to about 4.7 with concentratedhydrochloric acid and combined with about 800 liters of methylenechloride. The combined solvents are agitated for about 4 hours andseparated. The aqueous layer is combined with an additional 800 litersof methylene chloride and agitated for about 4 hours. The layers areseparated and each methylene chloride extract separately treated withabout 10 kilograms of Super-Cel and filtered. Both extracts areevaporated to a combined volume of about 60 liters.

PREPARATION 10

The 60 liter solution of C-076 in methylene chloride of the previousexample is concentrated to dryness in vacuo and the residue is combined3 times with 60 liter portions of methanol and evaporated to dryness toremove any residual methylene chloride. The final methanol concentratevolume is approximately 36 liters. The methanol solution is storedovernight and filtered. The filter cake is washed with 40 liters offresh methanol and the methanol filtrates and washings are combined. Themethanol solution is combined with 95 liters of ethylene glycol and 130liters of heptane. The 2 layer solution is agitated for 5 minutes andthe lower layer (ethylene glycol and methanol) is separated. The heptanesolution is washed with a mixture of 20 liters of ethylene glycol and6.3 liters methanol. After five minutes of agitation, the lower layer isseparated and combined with the first ethylene glycol/methanol extract.An equal volume of water (approximately 150 liters) containing 79 g. ofsalt per liter is added to the ethylene glycol/methanol extracts. Thissolution is extracted with 150 liters of ethyl ether with agitation for5 minutes. The ether layer is washed with 75 liters of water (1/2volume) and agitated for 5 minutes and the layers separated. Thisprocedure is repeated an additional 2 times (the final water washcontains 20 g. of salt per liter) affording a final ether layer volumeof 110 liters. The ether layer is concentrated in vacuo, to a minimumvolume, keeping the temperature less than 25° C. 40 Liters of methylenechloride is added to the residue and the solution is evaporated todryness. This procedure is repeated and the final residue concentratedin vacuo at 50° C. to dryness.

PREPARATION 11

A 30 centimeter diameter column is prepared with a layer of 34 kilogramsof activated alumina followed by a layer of 34 kilograms of activatedcarbon in a solution of methylene chloride. The residue from theprevious example is dissolved in methylene chloride to a volume of 34liters and applied to the column and eluted with 34 liters of methylenechloride, these fractions are discarded. A 3% solution of isopropanoland methylene chloride (20.8 liters of isopropanol and 660 liters ofmethylene chloride) is applied to the column and eluted in approximately200 liter fractions. The combined isopropanol and methylene chloridefractions are evaporated in vacuo at a bath temperature of about 60° C.to a volume of about 20 liters. The bath temperature is reduced to about45° C. and the extract is evaporated to dryness in vacuo. The residue isdissolved in 10 parts methylene chloride, 10 parts hexane and one partmethanol to a final volume of 15 liters. This solution is applieddirectly to the Sephadex LH-20 column of the next example.

PREPARATION 12

A 30 centimeter diameter column is prepared in methanol with 36kilograms of Sephadex LH-20 (available from Pharmacia Fine Chemicals,800 Centennial Avenue, Piscataway, N.J. 08854) and washed with a solventconsisting of 10 parts methylene chloride, 10 parts hexane and one partmethanol. One-fourth of the C-076 solution of Example 10 is applied tothe column and the column eluted at a rate of 250 ml. per minute. Two 20liter forecuts are collected and discarded followed by 20 two liter richcuts (identified as fractions 1-20), followed by a single 20 liter tailcut, which is discarded. Fractions 1-8 are found to contain the C-076 Acompounds and fractions 9-20 are found to contain the C-076 B compounds.

PREPARATION 13

The process of Preparation 12 is repeated on the same scale three moretimes and all of the fractions containing the C-076 B components(fractions 9-20) are combined and evaporated to dryness, affording 818g. of crude mixed C-076 B components. The sample is found to contain 55%C-076 B1 and 39% of C-076 B2. 680.5 G. of this sample is dissolved in 2liters of methylene chloride and placed in a 22 liter three neck roundbottom flask followed by the addition of 13.6 liters of methanol. Themethylene chloride is removed by distillation. 13.6 Liters of ethyleneglycol is added as the methanol is being distilled under reducedpressure. The rate of distillation is maintained such that thetemperature of the solution did not go below 65° C. When the addition ofthe ethylene glycol is complete, the solution is allowed to cool at 5°C. for sixteen hours. The crystals are filtered and washed with 1 literof cold ethylene glycol. The crystals are then redissolved in 2 litersof methylene chloride the solution placed in a 22 liter three neckedround bottom flask. The procedure described above is repeated twice. Thefirst time 12.5 liters each of methanol and ethylene glycol is employedand the second time 13.6 liters each of methanol and ethylene glycol isemployed. The final crystals are washed with 1 liter of cold ethyleneglycol and 1 liter of water. The crystals are dissolved in 4 liters ofwater and dried by filtering through sodium sulfate. The benzenesolution is concentrated to a volume of 2 liters and lyophilizedaffording 241.2 gm. of a white powder consisting of 98% C-076 B₁ and 1%of C-076 B₂.

The mother liquors (22 liters) from the first two crystallizations aboveare combined and diluted with 22 liters of water. The aqueous solutionis extracted with 60 liters of toluene and again with 15 liters oftoluene. The toluene extract is then washed with 48 liters of water. Theorganic phase is filtered through Super-Cel to remove any residual waterand evaporated affording 336 gm. of solid material consisting of 79%C-076 B₂ and 16% C-076 B₁ compounds.

PREPARATION 14

In the four Sephadex LH-20 columns of the procedure of Preparation 12,fractions 1-8 contain the C-076 A compounds and are combined. By HPLCanalysis the mixture is found to contain 252 g. of C-076 A2a, 16 g. ofA2b, 94 g. of A1a and 24 g. of A1b. The material is dissolved in asolvent system consisting of hexane:toluene:methanol in the proportionof 6:1:1 and applied to the Sephadex LH-20 column of the same dimensionsas the one used in Preparation 12 in the above solvent. Fractions arecollected at the rate of 250 ml. per minute and a 20 liter forecut iscollected and discarded. Further elution affords 2 additional 20 literforecuts which are also discarded and 50 four liter rich cuts whichcontain C-076 A compounds. Fractions 3-8 are found to containpredominately C-076 A1 components (40.2 g. A1a and 6.7 g. A1b), andfractions 29-36 are found to contain C-076 A2 compounds (117.2 g. A2aand 7.35 g. of A2b). Fractions 9-28 contain a mixture of C-076 A1 and A2compounds.

PREPARATION 15

A sample of 150 g. of C-076 B1 from Preparation 13 is dissolved in 3liters of a solvent mixture of hexane:toluene:methanol in the ratio of3:1:1. The solution is passed through a column of Sephadex LH-20 (of thesame dimensions as the one used in Preparation 12) in the above solventtaking fractions at the rate of 250 ml. per minutes. After two 20 literportions of the solvent mixture are collected and discarded, forecut of10 liters is taken and discarded. Then 30 richcuts of 2 liters each aretaken. Fractions 1-13 and 25-30 are discarded. Fractions 14-16 arecombined and contain 80 g. of predominately C-076 B1a. Fractions 22-24are combined and contain 6.7 g. of predominately C-076 B1 b. Fractions17-21 contain a mixture of C-076 B1a and B1b.

Fractions 17-21 above are combined and concentrated and passed through aSephadex LH-20 column with the same solvent system as above. Three 20liter forecuts are taken and discarded. Richcuts are then taken asfollows: 5 cuts of 2 liters each (fractions 1-5); 20 cuts of 1 litereach (fractions 6-25); and 10 cuts of 2 liters each (fractions 26-35).Fractions 1-15 are discarded; fractions 16-21 contain 13.5 g. of C-076B1 a and 0.4 g. of C-076 B1b; fractions 22-26 contain 44 g. of C-076 B1aand 0.13 g. of C-076 B1b; fractions 27-30 contain 10.2 g. of C-076 B1aand 0.8 g. of C-076 B1b.

PREPARATION 16

A mixture of all 8 C-076 components are chromatographed on a highpressure liquid chromatography column 4 mm.×30 cm. packed with 10 micronμ Bondapak C₁₈ silica gel (available from Waters Associates Inc., MapleStreet, Milford, Mass. 01757) eluting with 85:15 (v/v) methanol:water ata constant 40° C. At a flow rate of 1.2 ml. per minute all eightcompounds are separated and the elution volumes, which under theforegoing constant conditions are characteristic of the individualcompounds are as follows:

    ______________________________________                                                  Elution Volume (Ve) Ml                                              ______________________________________                                        C-076 B.sub.2 b                                                                           5.90                                                              C-076 B.sub.2 a                                                                           6.52                                                              C-076 A.sub.2 b                                                                           7.12                                                              C-076 A.sub.2 a                                                                           7.88                                                              C-076 B.sub.1 b                                                                           8.36                                                              C-076 B.sub.1 a                                                                           9.60                                                              C-076 A.sub.1 b                                                                           10.24                                                             C-076 A.sub.1 a                                                                           11.88                                                             ______________________________________                                    

The separation of C-076 "b" components from the respective "a"components is accomplished using techniques such as high pressure liquidchromatography. An absolute methanol solution of 30 microliters of amixture of C-076 A1a and A1b, estimated to contain 30 micrograms ofC-076 A1b is placed on a 3×250 mm. high pressure liquid chromatographycolumn containing Spherisorb 5 micron ODS (available from SpectraPhysics) as packing. The column is eluted with 85:15 methanol-water at arate of 0.15 ml./min. The elution of the products are followed byobserving the ultraviolet absorption of the eluent and collecting theindividual components at the outlet of the UV monitor. 30 Micrograms ofC-076 A1b is recovered in this manner.

What is claimed is:
 1. A compound having the formula: ##STR4## whereinthe broken line indicates a single or a double bond; R₁ is hydrogen orhalogen;R₂ is hydrogen, methyl or loweralkanoyl; R₃ is .[.n-propyl.]..Iadd.isopropyl .Iaddend.or sec-butyl; and R₄ is present only when thebroken line indicates a single bond and represents hydrogen, hydroxy,loweralkanoyloxy, loweralkylthio, loweralkylsulfinyl, loweralkylsulfonylor loweralkoxy; provided that when R₂ is hydrogen or methyl the brokenline can indicate only a single bond and R₄ is other than hydroxy. 2.The compound of claim 1 wherein R₃ is .[.n-propyl.]..Iadd.isopropyl.Iaddend..
 3. The compound of claim 1 wherein R₃ issec-butyl.
 4. The compound of claim 3 wherein the broken line indicatesa single bond and R₄ is hydrogen.
 5. The compound off claim 4 wherein R₁is chlorine, R₂ is hydrogen, R₃ is sec-butyl, R₄ is hydrogen, and thebroken line indicates a single bond, which is 13-chloro-13-deoxy-22,23,dihydro-C-076-B1a-aglycone.
 6. The compound of claim 4 wherein R₁ ishydrogen, R₂ is hydrogen, R₃ is sec-butyl, R₄ is hydrogen, and thebroken line indicates a single bond, which is13-deoxy-22,23-dihydro-C-076-B1a-aglycone.
 7. The compound of claim 4wherein R₁ is hydrogen, R₂ is methyl, R₃ is sec-butyl, R₄ is hydrogenand the broken line indicates a single bond, which is13-deoxy-22,23-dihydro-C-076-A1a-aglycone.
 8. The compound of claim 3wherein R₂ is a loweralkanoyl.
 9. The compound of claim 8 wherein R₁ ischlorine, R₂ is acetyl, R₃ is sec-butyl, and R₄ and the broken linerepresent a 22,23-double bond, which is 13-chloro-13-deoxy-C-076 B1aaglycone-5-O-acetate.
 10. The compound of claim 8 wherein R₁ ishydrogen, R₂ is acetyl, R₃ is sec-butyl and R₄ and the broken lineindicate a 22,23-double bond, which is13-deoxy-C-076-B1a-aglycone-5-O-acetate.
 11. The compound of claim 3wherein R₄ is loweralkanoyloxy.
 12. The compound of claim 11 wherein R₄is an acetoxy group.
 13. The compound of claim 12 wherein R₁ ischlorine, R₂ is hydrogen, R₃ is sec-butyl, R₄ is acetoxy, and the brokenline indicates a single bond, which is13-chloro-13-deoxy-C-076-B2a-aglycone 23-O-acetate.
 14. The compound ofclaim 12 wherein R₁ is hydrogen, R₂ is hydrogen, R₃ is sec-butyl, R₄ isacetoxy, and the broken line indicates a single bond, which is13-deoxy-C-076-B2a-aglycone 23-O-acetate.
 15. The compound of claim 3wherein R₂ is loweralkanoyl, and R₄ is loweralkanoyloxy.
 16. Thecompound of claim 15 wherein R₂ is acetate and R₄ is acetoxy.
 17. Thecompound of claim 16 wherein R₁ is chlorine, R₂ is acetyl R₃ issec-butyl, R₄ is acetoxy, and the broken line indicates a single bond,which is 13-chloro-13-deoxy-C-076-B2a-aglycone 5,23-di-O-acetate. 18.The compound of claim 16 wherein R₁ is hydrogen, R₂ is acetyl, R₃ issec-butyl, R₄ is acetoxy, and the broken line indicates a single bond,which is 13-deoxy-C-076-B2a-aglycone 5,23-di-O-acetate.